1. Technical Field
This invention relates to an enzymatic process for producing ribavirin.
The chemical nomenclature of ribavirin is 1-.beta.-D-ribofuranosyl-1,2,4-triazole-3-carboxamide, which is also known as virazole (trade name), and it is known as a compound having a broad and potent antiviral activity against DNA and RNA viruses [Annals of the New York Academy of Sciences 284, 272-292 (1977)].
2. Prior Art
The processes for producing ribavirin known in the art may be classified into synthetic methods, fermentative methods and enzymatic methods.
Representatives of synthetic methods known include methods in which 3-methoxycarbonyl-1,2,4-triazole is caused to react with 1-O-acetyl-2,3,5-tri-O-acyl-.beta.-D-ribofuranose, and the resultant 1-(2',3',5'-tri-O-acyl-.beta.-D-ribofuranosyl)-3-methoxy-carbonyl-1,2,4-tr iazole is treated with ammonia to effect amidation and deblocking thereof (see Japanese Patent Laid-open Nos. 4469/1973, 80070/1974 and 80071/1974); the method similar to the aforementioned in which an aralkyloxyl group is employed as the substituent at the 3-position of the triazole (see Japanese Laid-open Patent Application No. 160793/1980); methods in which 3-methoxycarbonyl-1,2,4-triazole is trimethylsilylated and caused to react with a halide of 2,3,5-tri-O-benzoyl-.beta.-D-ribofuranoside, followed by treatment with ammonia (see Japanese Patent Laid-Open Nos. 4469/1973 and 86372/1974); and others. In any of these synthetic methods, it would be required to block the active groups of the starting compounds before the reaction, or sometimes activation of the ribose may be required in carrying out the reaction. In some cases, heating to a high temperature would also be necessary. Further, after the reaction, deblocking and amidation must be performed, thus involving the problems of cumbersome reaction operations. As another problem, the positional selectivity during the condensation reaction is not high in any one of these methods as far as the present inventors are concerned.
Fermentative methods known in the art include methods in which 1,2,4-triazole-3-carboxamide is added at a time or intermittently to a culture medium containing carbon sources, nitrogen sources, inorganic substances and other nutrients necessary for cultivation of a microorganism employed before initiation of culturing or during culturing of a microorganism belonging to the genus Brevibacterium, the genus Corinebacterium, the genus Arthrobacter, the genus Micrococcus or the genus Bacillus, and cultivation is carried out for a period of time of 2 to 8 days after initiation of culturing, thereby to form and accumulate ribavirin in the culture medium [see Japanese Patent Publication No. 17830/1979; Journal of The Agricultural Chemical Society of Japan, 50 (9), 423-430 (1976)]. This method could involve following problems: (1) since production of ribavirin is carried out in a nutrient medium during proliferation of microorganisms, it is first necessary to prepare a culture medium containing various nutrient sources for proliferation of microorganisms, and cumbersome pre-treatments such as sterilization of the medium before inoculation of the seed microorganisms are also necessary; (2) cultivation for the purpose of accumulation of ribavirin, which is accompanied with proliferation of microorganisms, is carried out generally at a normal temperature of 20.degree. to 40.degree. C. and therefore it is constantly required to take care about contamination with miscellaneous microorganisms. Moreover, under such conditions, there also exists an activity for decomposing the ribavirin, thus lowering the yield of this objective compound; (3) proliferation of microorganisms must be carried out for a long period of time of 2 to 8 days; (4) various nucleosides, phosphorylated products of ribavirin and other metabolites are formed as by-products, and for recovery of ribavirin from the culture broth thus requires that ribavirin be separated not only from the starting compounds but also from various by-products, whereby isolation and purification are rendered cumbersome; and (5) microorganisms must be cultured each time in production of ribavirin.
Enzymatic methods known in the art include methods in which 1,2,4-triazole-3-carboxamide is caused to react with ribose-1-phosphate at a pH of 5-9 and at a temperature of 0.degree.-50.degree. C. in the presence of a nucleoside-phosphorylase [see Japanese Patent Laid-open No. 29720/1975]. The method could have problems that the ribose-1-phosphate used as a ribose donor is unstable and is not easily available, and that a preparation of the purified enzyme which is used is not easy to be prepared.